Antimicrobial wash and carrier solutions and uses thereof

ABSTRACT

Novel wash and carrier solutions ideally suited for topical administration (e.g., as wash solutions and as carrier solutions). The solutions described herein are non-toxic, non-irritating, isotonic, possess non-specific broad-spectrum antimicrobial properties, and have an alkaline pH. Also disclosed are sterile preparations of the solutions and methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/412,477, filed Apr. 11, 2003, and claims priority of U.S. ProvisionalApplication No. 60/371,755, filed Apr. 11, 2002, the disclosures of allof which are incorporated herein by reference in their entirety for allpurposes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the field of topical antimicrobial wash andcarrier solutions. The solutions disclosed herein are substantiallynon-irritating and thus, are especially suited to deliver topicallypharmaceutical active agents, to clean and/or treat wounds or tissues.

2. Description of the Related Art

A variety of antimicrobial wash and carrier solutions are currently usedto wash skin or other tissue surfaces, including wounds, to remove dirt,debris, or to loosen or soften crusted lymph or blood clots. In someinstances, such solutions are used to disinfect a tissue and thus, curbor prevent an infection. In addition, such solutions can be used ascarriers for pharmaceutically active agents to be topically applied to agiven tissue.

Under normal circumstances (i.e., in the absence of heightened painsensitivity) a wash solution should preferably be non-irritating. Thetissues treated with wash solutions are often inflamed and thus, have aheightened sensitivity to pain, compelling the use of non-irritatingsubstances. Unfortunately, a great number of wash solutions presentlyavailable elicit tissue irritation, making their use in the pediatricand veterinary setting cumbersome. For the most part, non-specificdisinfectant/antimicrobial solutions (e.g., solutions including aceticacid) described in the art may actually cause or exacerbate tissueirritation at the site of use because of their nature and/or effectiveconcentration or treatment time required.

Solutions, which differ from normal serum in tonicity, have been shownto cause pain on injection, electrolyte shifts, or even tissueirritation following contact. This effect depends on the degree ofdeviation from isotonicity. For example, ophthalmic preparations forinstillation in the eye should be isotonic in nature to avoidirritation. Therefore, wash solutions should be isotonic in nature toavoid exacerbation of irritation associated with tissue damage (seee.g., Remington: The Science and Practice of Pharmacy, 19^(th) Ed., Ch.,36, Gennaro, A. R., Ed, Mack Publishing Co. (1995)).

Wash solutions have been used as carrier solutions by supplementingthem, for example, with one or more antibiotic(s). Moreover,combinations of Tris-EDTA solutions with specific antibiotics have beenpostulated to have a synergistic effect against a variety of infectiousagents and have been described extensively in the literature at least asearly as 1974 (see for example Blue et al. (1974), Wooley et al. (1983),Farca et al. (1997), see also PCT/US01/29133 describing variousformulations for the treatment of specific Gram positive and Gramnegative bacterial infections using Tris-EDTA, a variety of antibiotics,and a carrier). However, quite often the effectiveness of thiswell-known approach is hampered by the development of microbialantibiotic resistance.

An ideal wash/carrier solution should also be a non-specificbroad-spectrum antimicrobial for initial use until the specificinfection is identified, and subsequently as a carrier (e.g., forantibiotics). Non-specific broad-spectrum antimicrobial properties areuseful (a) to aid in cleansing and treating the underlying tissue (e.g.,to treat an existing infection or prevent an infection); and (b) tominimize and preferably even prevent the occurrence of backcontamination of a multi-dose dispenser, thereby minimizing inadvertentcross-contamination from use to use and/or from user to user.

Several solutions described in the literature include a rather harshdisinfecting agent such as boric acid which are known to be toxic (boricacid has been removed from many hospitals after reports showed thatrepeated applications to damaged tissues resulted in the absorption ofboric acid in sufficient amounts to cause acute poisoning causing deathin half of the patients accidentally intoxicated (Goodman and Gilman'sThe Pharmacological Basis of Therapeutics, 5^(th) Ed., Ch. 50, page 994,McGraw Hill Companies Inc., New York (1975)). To exemplify, U.S. Pat.No. 5,480,658 describes a “pH balanced”—e.g., alkaline pH—multi-purposecleaning solution containing acetic acid and boric acid in a water basefor the routine cleaning of the ear, the prevention and treatment of eardisease such as “swimmer's ear,” and wound cleaning. Similarly, U.S.Pat. No. 5,853,767 discloses a solution comprising, as activeingredients, both acetic acid and boric acid for the treatment ofbacterial and fungal skin infections. The solution is described ashaving bacteriostatic, bacteriocidal and anti-fungal properties, and asuseful for the treatment of vaginal infections, such as vaginitis.

The alkaline pH is useful for conditioning and softening biologicaltissues, and increasing the penetration of pharmaceutical agents. Theexudates associated with infected lesions are acidic in nature and caninactivate or reduce the efficacy of many commonly used antibioticagents. An alkaline pH wash solution can aid in the neutralization ofthe acidic environment of the lesion thereby allowing for increasedefficacy for the commonly used antibiotic agents. Unfortunately, severalsolutions described to date are acidic. U.S. Pat. No. 5,631,218 is to adisinfecting solution including an ortho-hydroxy benzoic acid derivative(e.g., salicylic acid) and an amphoteric surfactant and/or analkoxylated alcohol nonionic surfactant (e.g., an ethoxylated alcohol).The solution has a pH of 1.0-5.5. Similarly, U.S. Pat. No. 6,214,363discloses a rinse-off antimicrobial cleansing composition including anantimicrobial agent, an anionic surfactant, a proton donating agent, anda deposition aid in water, in which the composition is adjusted to a pHof from about 3.0 to about 6.0.

Other solutions described in the art include U.S. Pat. No. 6,211,238disclosing anionic surfactants that are used in conjunction with anantifungal acid and a chelating agent to preserve topicallyadministrable pharmaceutical compositions without the need for aconventional preservative, such as benzalkonium chloride. U.S. Pat. No.6,284,749 discloses a preservative system for topically administrablepharmaceutical compositions including fatty acid/amino acid soaps inconjunction with an antifungal acid and a chelating agent.

Thus, there is a need for wash and carrier solutions overcoming theshortcomings of presently available solutions. Such wash and carriersolutions ideally should be non-toxic, non-irritating, isotonic, possessnon-specific broad-spectrum antimicrobial properties, and have analkaline pH.

SUMMARY OF THE INVENTION

The inventors have devised novel wash and carrier solutions ideallysuited for topical administration (e.g., as wash solutions and ascarrier solutions). The solutions described herein are non-toxic,non-irritating, isotonic, possess non-specific broad-spectrumantimicrobial properties, and have an alkaline pH.

Thus, in one aspect, the present invention provides a non-irritatingaqueous antimicrobial and preservative wash and carrier solution, foruse as a wash solution for biological tissues or a carrier solution fordelivering pharmaceuticals to biological tissues, including (a) a buffersystem for maintaining the solution at an alkaline pH, (b) a surfactantsystem, (c) a metal ion chelating agent, and (d) a non-ionicpreservative having antimicrobial activity.

In some embodiments, the buffer system is a Tris buffer system, aphosphate buffer system, or a citrate buffer system. In certainembodiments, the buffer system is a Tris buffer system includingtromethamine base at a concentration of about 0.1-2.0 (w/w) andtromethamine hydrochloride at a concentration of about 0.1-2.0 (w/w). Inan embodiment the Tris buffer system according to the invention includestromethamine base at a concentration of about 0.45-0.55 (w/w). In yetanother embodiment, the Tris buffer system according to the inventionincludes tromethamine hydrochloride at a concentration of about 0.3-0.45(w/w). In another embodiment, the Tris buffer system according to theinvention includes tromethamine base at a concentration of about 0.465%(w/w) and tromethamine hydrochloride at a concentration of about 0.43%(w/w).

In some embodiments, the buffer system has a pH from about 7.5 to about9.5, or a pH from about 8 to about 9, or a pH from about 8.2 to about8.8. In one embodiment, the buffer system of the invention has a pH fromabout 8.4 to about 8.6.

In certain embodiments, the surfactant is a non-ionic surfactant. Insome embodiments, the surfactant includes a multiplicity of etherlinkages. Non limiting surfactants according to the invention, includewithout limitation, nonoxynol 12, PPG-12/PEG-50 Lanolin, or phospholipidCDM. When the surfactant is nonoxynol 12, it is present at aconcentration of up to about 2.0% (w/w). In certain embodiments,nonoxynol 12 is present at a concentration of about 0.3-0.8%, mostpreferably of about 0.5% (w/w). When the surfactant is PPG-12/PEG-50lanolin, it is present at a concentration of up to about 2.0% (w/w). Incertain embodiments, PPG-12/PEG-50 lanolin is present at a concentrationof about 0.3-0.8%, most preferably of about 0.5% (w/w). When thesurfactant is phospholipid CDM, it is present at a concentration fromabout 0.2 to about 3% (w/w). In other embodiments, phospholipid CDM ispresent at a concentration of about 2% (w/w).

Wash solutions according to the instant invention include at least onemetal ion chelating agent. Chelating agents are well known in the art.Non-limiting representative chelating agents suitable according to theinvention include citric acid; phosphates; disodium EDTA; tetrasodiumEDTA; ethylene glycol-bis-(b-aminoethylether)-N,N,N′,N′-tetraacetic acid(EGTA); 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid(BAPTA); ethylene-N,N′-diglycine (EDDA); 2,2′-(ethylendiimino)-dibutyricacid (EBDA); lauroyl EDTA; dilauroyl EDTA, triethylene tetraminedihydrochloride (TRIEN), diethylenetriamin-pentaacetic acid (DPTA),triethylenetetramine hexaacetic acid (TTG), deferoxamine, Dimercaprol,zinc citrate, penicilamine succimer, Editronate, and edetate calciumdisodium.

In particular embodiments, the metal ion chelating agent is tetrasodiumEDTA at a concentration of from about 0.05% (w/w) to about 0.5% (w/w).In some embodiments, tetrasodium EDTA is present at a concentration offrom about 0.1% (w/w) to about 0.15% (w/w). In another embodiment,tetrasodium EDTA is present at a concentration of about 0.126% (w/w).

Some wash solutions according to the invention may optionally include apreservative. Many preservative agents are well know to one of skill inthe art. Preservatives which may be included according to the invention,include without limitation, imidazolindyl urea, benzalkonium chloride,diazolindinyl urea, dimethylol dimethylhydantoin, mixtures of5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one(also known as KATHON CG®), benzyl alcohol, phenylethyl alcohol,phospholid CDM, and chlorobutanol, parabens, benzoic acid, and sorbicacid.

In certain embodiments, the preservative is benzyl alcohol at aconcentration of up to about 2% (w/w). In some embodiments, benzylalcohol is present at a concentration of about 0.5-1.5% (w/w). In anembodiment, benzyl alcohol is present at a concentration of about 1.2%(w/w). In certain embodiments, the preservative is benzyl alcohol at aconcentration of up to about 2% (w/w). In some embodiments, KATHON CG®is present at a concentration of up to 0.1% (w/w). In other embodiments,KATHON CG® is present at a concentration of about 0.05% (w/w).

In some embodiments, the tonicity of the solution is about 150-350mOsmol, or about 200-300 mOsmol.

In a second aspect, the invention provides methods of using thesolutions of the invention according to the first aspect for washingwounds, treating a topical microbial infection, and topically deliveringa pharmaceutical agent to a tissue. The topical microbial infection mayinclude, for example, a Pseudomonas infection of the ear or ear canalcausing otitis externa.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides wash and carrier solutions ideally suitedfor topical administration as wash solutions for cleaning biologicaltissues and as carrier solutions for delivering pharmaceuticals tobiological tissues. The solutions described herein are non-toxic,non-irritating, isotonic, possess non-specific broad-spectrumantimicrobial properties, and have an alkaline pH.

The solutions may be used, for example, to clean external surfaces toremove infectious agents or parasites at wound sites or to preparetissue surfaces prior to surgeries. Thus, for example, the solutions maybe used to wash biological tissues, such as bodily surfaces or orificeswhere there are infections or infestations of parasites (e.g., mammalianears or ear canals infected or infested with mites or bacteria), orwounds where infectious agents may be present (e.g., bacterial or viralagents, which can enter the body through an open wound). Similarly, thesolutions may be used to deliver pharmaceutically active agents totissues by serving as biocompatible carriers which are chemicallyinactive with respect to the pharmaceutically active agent (e.g.,antibiotics, antifungals). Because the solutions contain surfactants,they can be used as a solvent for pharmaceuticals that have limitedsolubility in aqueous solution and, therefore, are commerciallydistributed in solid or powder form.

The patent, scientific and medical publications referred to hereinestablish knowledge that was available to those of ordinary skill in theart at the time the invention was made. The patents and scientificliterature referred to herein establish the knowledge of those withskill in the art and are hereby incorporated by reference in theirentirety to the same extent as if each was specifically and individuallyindicated to be incorporated by reference. Any conflict between anyreference cited herein and the specific teachings of this specificationshall be resolved in favor of the latter. Likewise, any conflict betweenan art-understood definition of a word or phrase and a definition of theword or phrase as specifically taught in this specification shall beresolved in favor of the latter.

All technical and scientific terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art; references to techniques employedherein are intended to refer to the techniques as commonly understood inthe art, including variations on those techniques or substitutions ofequivalent techniques which would be apparent to one of skill in theart.

Technical and scientific terms used herein have the meaning commonlyunderstood by one of skill in the art to which the present inventionpertains, unless otherwise defined. Reference is made herein to variousmethodologies and materials known to those of skill in the art. Standardreference works setting forth the general principles of formulationchemistry include Pharmaceutical Dosage Forms, 2^(nd) Ed., MarcelDekker, Inc., New York, (1990). Standard reference works setting forththe general principles of pharmacology include Goodman and Gilman's ThePharmacological Basis of Therapeutics, 10^(th) Ed., McGraw HillCompanies Inc., New York (2001). Any suitable materials and/or methodsknown to those of skill can be utilized in carrying out the presentinvention. However, preferred materials and methods are described.Materials, reagents, and the like to which reference are made in thefollowing description and examples are obtainable from commercialsources, unless otherwise noted.

As used in this specification, the singular forms “a,” “an” and “the”specifically also encompass the plural forms of the terms to which theyrefer, unless the content clearly dictates otherwise. For example,reference to “surfactant” includes mixtures of surfactants.

As used in this specification, whether in a transitional phrase or inthe body of the claim, the terms “comprise(s)” and “comprising” are tobe interpreted as having an open-ended meaning. That is, the terms areto be interpreted synonymously with the phrases “having at least” or“including at least”. When used in the context of a process, the term“comprising” means that the process includes at least the recited steps,but may include additional steps. When used in the context of a compoundor composition, the term “comprising” means that the compound orcomposition includes at least the recited features or components, butmay also include additional features or components.

In order to more clearly and concisely describe the subject matter whichis the invention, the following definitions are provided for certainterms which are used in the specification and appended claims.

As used herein, the term “about” means approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20%.

In a first aspect the invention provides aqueous antimicrobial andpreservative solutions which include a buffer system for maintaining thesolution at an alkaline pH, a surfactant system, a metal ion chelatingagent, and a non-ionic preservative having antimicrobial activity.Solutions according to this first aspect are non-irritating.

Buffer Systems

The use of an alkaline pH solution offers the advantage of increasingthe transmembrane distribution and penetration of some drugs across thecell membrane. Most drugs are either weak acids or bases that formionized and nonionized species in solution. The nonionized species areusually lipid soluble and are able to penetrate the lipid membrane of acell whereas the ionized species have difficulty in crossing the cellmembrane. At steady state, the acidic species will accumulate on themore basic side of the membrane while basic species tend to accumulateon the acidic side of the membrane. The alkaline nature of the washsolution of the instant invention should produce an external environmentwherein the basic, ionized species of drugs should accumulate to theinterior of the cell membrane. This occurs as a purely physical processthat does not require active transport. Goodman and Gilman's ThePharmacological Basis of Therapeutics, 10^(th) Ed., Ch. 1., McGraw HillCompanies Inc., New York (2001). The exudates associated with infectedlesions are acidic in nature and can inactivate or reduce the efficacyof many commonly used antibiotic agents. An alkaline pH wash solutioncan aid in the neutralization of the acidic environment of the lesionthereby allowing for increased efficacy for the commonly used antibioticagents.

A characteristic of the solutions described lies in the buffer systemselected to maintain the solution at an alkaline pH. As used herein,“alkaline pH” means a pH greater than 7.0. In some embodiments, thebuffer solution is chosen to maintain the pH of the solution in therange of about 7.5 to about 9.5, or of about 8 to about 9, or of about8.2 to about 8.8. In another embodiment, the buffer system of theinvention maintains a pH from about 8.4 to about 8.6.

Buffer systems suitable for use in the present invention include any ofthose which are known in the art, or may hereafter be developed, andwhich can maintain the pH of the solution in the prescribed range for asubstantial period of storage (e.g., two months storage at 40° C.).Suitable buffer systems include Tris buffer systems, phosphate buffersystems, citrate buffer systems, and any other basic amino acid buffersystems.

As used herein, the term “Tris buffer system” means an admixture in asolution of Tris base and hydrochloride in molar ratios which act tostabilize the pH of the solution. Tris is also known as tromethamine ortrimethylol aminomethane or 2-amino-2-(hydroxymethyl)-1,3-propane-diol.

In some embodiments, the buffer system is a Tris buffer system, aphosphate buffer system, or a citrate buffer system. In certainembodiments, the buffer system is a Tris buffer system includingtromethamine base at a concentration of about 0.1-2.0 (w/w) andtromethamine hydrochloride at a concentration of about 0.1-2.0 (w/w). Inanother embodiment the Tris buffer system according to the inventionincludes tromethamine base at a concentration of about 0.45-0.55 (w/w).In other embodiments, the Tris buffer system according to the inventionincludes tromethamine hydrochloride at a concentration of about 0.3-0.45(w/w). In yet another embodiment, the Tris buffer system according tothe invention includes tromethamine base at a concentration of about0.465% (w/w) and tromethamine hydrochloride at a concentration of about0.43% (w/w). Tris buffers are commercially available as, e.g., fromSigma Chemical Co., St. Louis, Mo.

As used herein, the term “phosphate buffer system” means an admixture ina solution of monosodium phosphate, disodium phosphate and/or trisodiumphosphate in molar ratios which act to stabilize the pH of the solution.

As used herein, the term “citrate buffer system” means an admixture in asolution of citric acid and sodium hydroxide or citric acid andtrisodium citrate or other equivalents in molar ratios which act tostabilize the pH of the solution.

Surfactants

In another aspect, the solutions of the present invention include asurfactant. As used herein, “surfactant” means a soluble compound thatreduces the surface tension of liquids, or reduces interfacial tensionbetween two liquids or a liquid and a solid. Surfactants are typicallyamphipathic compounds, such as detergents, having a polar and anon-polar group.

The surfactant is useful for removing or solubilizing oily, fatty, orwaxy materials from the surface of a tissue. For example, by removing alayer of oil from skin, a pharmaceutical preparation applied topicallyto the skin can have more direct contact to the tissue and thereby bemore effective. The surfactant can also increase the usefulness of thesolution as a carrier by increasing the solubility of hydrophobicpharmaceuticals within the aqueous solution.

In some embodiments, the surfactant is a non-ionic surfactant. Non-ionicsurfactants may be preferred because they cause less irritation tobiological tissues. In some embodiments, the surfactant includes amultiplicity of ether linkages. Surfactants with ether linkages betweenthe polar group and the non-polar group may be preferred because theyare relatively more stable than ester linkages.

In some embodiments, the surfactant is at least one of nonoxynol 12(Jeen International, Little Falls, N.J.), PPG-12/PEG-50 lanolin (JeenInternational, Little Falls, N.J.), or phospholipid CDM, phospholipidEFA, phospholipid PTA (Uniquema, Paterson, N.J.) PEG-100 stearate, othernonoxynol or octoxynol surfactants with different PEG chain lengths. Inother embodiments, the solution may contain two or more surfactants.

When the surfactant is nonoxynol 12, it is present at a concentration ofup to about 2.0% (w/w). In some embodiments, nonoxynol 12 is present ata concentration of about 0.3-0.8%, most preferably of about 0.5% (w/w).When the surfactant is PPG-12/PEG-50 lanolin, it is present at aconcentration of up to about 2.0% (w/w). In certain embodiments,PPG-12/PEG-50 lanolin is present at a concentration of about 0.3-0.8%,most preferably of about 0.5% (w/w). When the surfactant is phospholipidCDM, it is present at a concentration from about 0.2 to about 3% (w/w).In certain embodiments, phospholipid CDM is present at a concentrationof about 2% (w/w). Phospholipid CDM is coco PG-dimonium chloridephosphate, a synthetic phospholipid surfactant.

One of skill in the art will appreciate that more than one surfactantmay be included. Hence, in representative examples provided hereinafter,both nonoxynol 12 and PPG-12/PEG-50 lanolin are included.

Metal Ion Chelating Agents

In another aspect, the solutions of the present invention include ametal ion chelating agent. As used herein, the term “metal ion chelatingagent” or simply, “chelating agent” means a chemical capable tosequester metal ions (such as for example magnesium and calcium ions)from a chemical or biological reaction mixture or solution by bindingtightly to the metal ions thereby preventing them from participating inother chemical or biological reactions. The chelating agent is useful toremove magnesium and calcium ions from a solution or from the surface ofa wound or tissue. Metal chelating agent can be useful in limiting thesurvival, growth or reproduction of microbes.

For example, a chelating agent can be used to chelate membrane-boundcalcium and magnesium in Gram-negative bacilli (e.g., Pseudomonasspecies), weakening the bacterial membranes and thereby increasing thepermeability of the bacterial membranes to antibiotics. Alternatively,or in addition, chelating agents such as EDTA (ethylene diamine tetraacetate (commercially available from various vendors including asVERSENE 100™ from Dow Chemical Co., Midland, Mich.)), can bind to andinactivate elastase on the surface of bacteria such as Pseudomonasspecies. Because the action of elastase is believed to be involved inthe virulence of some bacteria by allowing them to dissolve the tightjunctions between a host's epithelial cells, the chelating agent couldreduce the virulence of such bacteria and be used in the treatment ofsuch infections (e.g., otitis externa due to Pseudomonas infection).

Chelating agents are well known in the art. Acceptable chelating agentsuseful in the present invention include amino carboxylic acid compoundsand pharmaceutically acceptable salts thereof. Non-limitingrepresentative chelating agents suitable according to the inventioninclude citric acid; phosphates; disodium EDTA; tetrasodium EDTA;ethylene glycol-bis-(b-aminoethylether)-N,N,N′,N′-tetraacetic acid(EGTA); 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid(BAPTA); ethylene-N,N′-diglycine (EDDA); 2,2′-(ethylendiimino)-dibutyricacid (EBDA); lauroyl EDTA; dilauroyl EDTA, triethylene tetraminedihydrochloride (TRIEN), diethylenetriamin-pentaacetic acid (DPTA),triethylenetetramine hexaacetic acid (TTG), deferoxamine, Dimercaprol,zinc citrate, penicilamine succimer, Editronate, and edetate calciumdisodium.

In particular embodiments, the metal ion chelating agent is tetrasodiumEDTA at a concentration of from about 0.05% (w/w) to about 1.0% (w/w).In other embodiments tetrasodium EDTA is present at a concentration offrom about 0.1% (w/w) to about 0.15% (w/w). In another embodiment,tetrasodium EDTA is present at a concentration of about 0.126% (w/w).

Preservatives

Some wash solutions according to the invention include a preservativewith antimicrobial activity. As used herein, the term “preservative”means a compound that inhibits microbial growth, has antimicrobialactivity, or otherwise inhibits the deterioration of a solution to whichit is added. In some embodiments the preservative prevents the growth ofmicrobes in the solution (to minimize or prevent back contamination fromuse to use and/or from user to user of a multidose dispenser filled withthe solutions) and provide antimicrobial activity against microbespresent at a wound or tissue.

Many preservative agents are well known to one of skill in the art.Preservatives which may be included according to the invention, includewithout limitation, imidazolindyl urea, benzalkonium chloride,diazolindinyl urea, dimethylol, mixtures of5-chloro-2-methyl-4-isothiazolin-3-one (1.5% w/w) and2-methyl-4-isothiazolin-3-one (0.35% w/w) (commercially available asKATHON CG® from Rohm Haas, Bayport, Tex.) dimethylhydantoin, benzylalcohol (Velsicol Chemical, Rosemont, Ill.) phenylethyl alcohol, orchlorobutanol, parabens, benzoic acid, and sorbic acid. Also known inthe art, surfactants may display antimicrobial preservative properties,e.g. phospholipid CDM.

As is known in the art, quaternary amines are also generally useful aspreservatives. Non-limiting examples of quaternary amines includeQuaternium-15 (Dow Chemical, Midland, Tex.), cetrimide andcetylpridinium chloride (Zeeland Chemicals, Zeeland, Mich.),imidazolidinyl urea (available as Germall 115, Sutton Labs, Chatham,N.J.), benzalkonium chloride (Stepan Co., Northfield, Ill.), andbenzethonium chloride (Aldrich, Milwaukee, Wis.).

In certain embodiments, the preservative is benzyl alcohol at aconcentration of up to about 2% (w/w). In some embodiments, benzylalcohol is present at a concentration of about 0.5-1.5% (w/w). In anembodiment, benzyl alcohol is present at a concentration of about 1.2%(w/w). In certain embodiments, the preservative is benzyl alcohol at aconcentration of up to about 2% (w/w). In some embodiment, KATHON CG® ispresent at a concentration of up to 0.1% (w/w). In another embodiment,KATHON CG® is present at a concentration of about 0.05% (w/w).

Preservatives according to the invention are either non-irritating bytheir nature or are present at a non-irritating concentration to normalor damaged tissues. Hence, for example benzyl alcohol, by its verynature is a potential irritant. However, it was unexpectedly found that(contrary to the teachings in the literature see Handbook ofPharmaceutical Excipients, 2^(nd) Ed., Wade, A., and Weller, P., ThePharmaceutical Press, London GB, 1994, page 35-37) benzyl alcoholpresent at a non-irritating concentration is an antimicrobialpreservative at an alkaline pH.

As used herein, “irritation” or “irritating” refers to a higher thannormal sensitivity to external stimuli. In some instances, the tissuebeing treated may be inflamed and thus may present a heightenedsensitivity or excitability such that the application of an ordinarystimulus produces pain or excessive action or reaction. “Non-irritating”means that the application of stimulus results in minimal pain orreaction.

In order to further minimize the potential for irritation, the solutionsof the invention approach isotonicity. Solutions, which differ fromnormal serum in tonicity, have been shown to cause pain on injection,electrolyte shifts, or even tissue irritation following contact. Thiseffect depends on the degree of deviation from isotonicity. For example,ophthalmic preparations for instillation in the eye should be isotonicin nature to avoid irritation. Therefore, wash solutions should beisotonic in nature to avoid exacerbation of irritation associated withtissue damage (see e.g., Remington: The Science and Practice ofPharmacy, 19^(th) Ed., Chapt. 36, Gennaro, A. R., Ed, Mack PublishingCo. (1995)).

The tonicity of the solutions of the invention are about 150-350 mOsmol,or about 200 mM-300 mOsmol to reduce the likelihood or degree ofirritation. As used herein, “isotonicity” means having the sameconcentration of solutes as found in blood. As such, an isotonicsolution produces neither a net gain nor loss of water into cellsexposed to such solution.

The solutions described herein may be dispensed from any dispenser asnecessary to meet the specific contingencies. Those of skill willappreciate that the choice of dispenser size, shape and material may bedictated by a variety of parameters including the chemical nature of thecomponents. For example, where benzyl alcohol is the preservative, highdensity polyethylene (HDPE) materials should be used.

In order to reduce the likelihood of infection of biological tissues,the solution may be prepared in a sterile environment and be maintainedas a sterile solution until use. Hence, the invention provides a sterilepreparation of antimicrobial wash and carrier solution as describedabove in a sterile airtight container. Such sterile preparations may beused in any of the methods described below.

In a second aspect, the invention provides methods of using thesolutions of the invention according to the first aspect for washingwounds, treating a topical microbial infection, and topically deliveringa pharmaceutical agent to a tissue.

The solutions, preparations, and methods may be used with any animaltissue, preferably any mammalian tissues including, but not limited to,human, non-human primate, equine, bovine, ovine, porcine, canine andfeline tissues. The solutions may, for example, be used for thetreatment of infections and infestations of the ear and ear canal inmammals, such as humans and dogs.

In another aspect, the invention provides a method of washing a woundusing an antimicrobial wash and carrier solution as described above. Thewound may be one that is known to be infected with microbes orsusceptible to infection with microbes. The scope of the presentinvention contemplates the use of the solutions as a mouth rinse toinhibit the growth of infective lesions in the oral cavity, or as animmersion bath for treating superficial infective lesions of the skin(whole body) or an individual limb.

In another aspect, the invention provides a method of treating a topicalmicrobial infection of a tissue by washing the tissue with anantimicrobial wash and carrier solution as described above. The topicalmicrobial infection may include, for example, a Pseudomonas infection ofthe ear or ear canal causing otitis externa.

In another aspect, the invention provides a method of topicallydelivering a pharmaceutically active agent or medicament to a tissue bymixing the pharmaceutical agent with an antimicrobial wash and carriersolution as described above, and applying the resulting mixture to thetissue.

As used herein, “pharmaceutically active agent” or “medicament” shallrefer to ophthalmic, dermatological, otic or nasal agents that can betopically applied. Preferred pharmaceutically active agents includeantimicrobial agents (including antiviral agents, antifungal agents andbacteriocidal and bacteriostatic agents). Antibiotics are especiallycontemplated as pharmaceutically active agents in light of reportedevidence of synergistic effects when using combinations of Tris-EDTAsolutions with specific antibiotics (see for example Blue et al. (1974),Wooley et al. (1983), Farca et al. (1997), see also PCT/US01/29133).

Further non-limiting examples of pharmaceutically active agentsaccording to this aspect of the invention include: muscarinics (e.g.,pilocarpine), beta-blockers (e.g., betaxolol and timolol), anti-glaucomaagents, dopaminergic agonists and antagonists, prostaglandins, carbonicanhydrase inhibitors (e.g., acetazolamide, methazolamide andethoxzolamide), non-steroidal and steroidal anti-inflammatories, such assuprofen, ketorolac, dexannethasone, rimexolone and tetrahydrocortisol,alpha adrenergic receptor agonists (e.g. para-amino clonidine,brimonidine), proteins, growth factors, anti-infective such asciprofloxacin, and anti-allergic agents, (e.g. cromolyn sodium,emedastine and olopatadine). The invention also contemplatescombinations of two or more pharmaceutically active agents.

Additionally, the formulations of the invention may include otheroptional ingredients such as for example, antioxidants, pharmaceuticallyacceptable buffers or excipients, pH adjusting or viscosity modifying ortonicity agents, drug carriers, sustained-release agents,comfort-enhancing agents, solubilizing aids, and other stabilizingagents.

In any of the foregoing methods, the solution may be applied by anystandard means known to medical or veterinary professionals including,but not limited to, application by moistened swab, moistened cotton,moistened gauze, polymer foam, medically acceptable sponge or othercloth, syringe, squeeze bottle, dropper or pipette. Additionally, thesolutions of the invention may be applied using gels or membrane films.The invention also contemplates the use of application methods andmaterials that are acceptable for oral administration into the mouth ofthe individual. The solution can also be poured directly onto the tissuefrom an open vessel or container, or can be placed into a tank orreservoir from which it is dispensed through a hose or nozzle, with orwithout added pressure. Application of the solutions under pressurethrough a hose or nozzle can be particularly useful for the irrigationand cleansing of tissues and wound sites.

The following examples are intended to further illustrate certainembodiments of the invention and are not limiting in nature. Thoseskilled in the art will recognize, or be able to ascertain, using nomore than routine experimentation, numerous equivalents to the specificsubstances and procedures described herein. Such equivalents areconsidered to be within the scope of this invention, and are covered bythe following claims.

Examples Example 1 Antimicrobial Wash and Carrier Solutions

An antimicrobial wash and carrier solution according to the inventionwas made by standard methodologies familiar to any person of skill inthe chemical arts. Generally, the steps are: (a) add the chelating agent(e.g., tetrasodium EDTA) to a beaker of H₂O while stirring tosolubilize; (b) add the buffer system (e.g., tromoethamine base and thetromoethamine HCl) while stirring to dissolve; (c) add the preservative(e.g., benzyl alcohol) while stirring; and (d) add surfactant (e.g.,nonoxynol 12) while stirring; and (e) adjust the pH to the desired range(e.g., 7.5 to 9.5); finally (f) add H₂O to the desired final volume.

Preferred % Range Ingredient Amount (mg/g) (w/w) Tromethamine Base 46.50.3-0.6% Tromethamine Hydrochloride 43.0 0.3-0.6% Tetrasodium EDTA 12.60.05-0.2%  Nonoxynol 12 50 0.3-0.8% PPG-12/PEG-50 Lanolin 50 0.3-0.8%Benzyl alcohol 120 0.5-2.0% Water Purified, USP 9677.9 —

In this example, the tromethane (Tris) base and hydrochloride serve asthe buffer system, the tetrasodium EDTA is the metal ion chelatingagent, the nonoxynol 12 and PPG-12/PEG-50 Lanolin are both surfactants,and the benzyl alcohol is the preservative with antimicrobial activity.

Example 2 Additional Antimicrobial Wash and Carrier Solutions

Additional antimicrobial wash and carrier solutions according to theinvention were prepared by the same protocol as described above toinclude the following materials:

FORMULATION ID # 940-16C 940-6a 940-8b 940-12D 940-14F Tromethamine Base4.7 4.6 5.4 4.6 4.6 Tromethamine Hydrochloride 4.3 4.0 3.3 4.3 4.3Tetrasodium Edetate 1.3 1.2 1.2 1.3 1.3 Nonoxynol 12 5 0 0 0 1PPG-12/PEG-50 Lanolin 5 0 0 0 5 Phospholipid CDM 0 20 20 20 0 Benzylalcohol 12 0 0 0 12 Kathon CG ® 0 0.5 0 0 0 Water Purified, USP QS AD1000 1000 1000 1000 1000 pH 8.5 8.0 8.6 8.2 8.1

Example 3 Antimicrobial Effectiveness of Benzyl Alcohol

Various concentrations of benzyl alcohol were tested for antimicrobialpreservative effectiveness (APE) using the USP 25 test 51 (U.S.Pharmacopeia 25, U.S. Pharmacopeia, MD (2001)). The formulations beloware based on the formulation designated as 940-16 C (with a slightlyhigher pH) in Example 2 above with varying benzyl alcoholconcentrations. The table below summarizes the results of these studies.

Sample ID Benzyl alcohol pH APE results 940-19A 1.20% w/w 8.60 Pass940-19B 0.96% w/w 8.60 Pass 940-19C 0.70% w/w 8.68 Pass (*) see Example2

These results are surprising in that the prior art teaches that benzylalcohol does not have significant preservative activity at a pH above8.0. Indeed, it is generally taught in the art that many preservativesare not effective at alkaline pH. For example, sorbic acid and salicylicacid are active as antimicrobial preservatives only in the acidic pHrange of about 4.0-6.0. Similarly, benzoyl alcohol, 2-phenoxyethanol,and benzoic acid are typically used only in the acidic pH range of about4.0-5.0 and are not effective at a pH above 8.0 (see, Handbook ofPharmaceutical Excipients (2nd edition, 1994), A. Wade and P. J. Weller,eds., pg. 35). However, as the above data demonstrate, benzyl alcohol atpH 8.5 meets the USP antimicrobial preservative requirement.

A comparison study shows that, when the various concentrations of benzylalcohol are tested for antimicrobial preservative effectiveness (APE),the addition of benzyl alcohol produces both enhanced antimicrobialactivity and prolonged shelf life as compared to solutions of theinstant invention lacking benzyl alcohol.

Antimicrobial activity can be measured by any standard assay known tothose of skill in the art (e.g., USP 25 antimicrobial preservativeeffectiveness (APE) test 51 (U.S. Pharmacopeia 25, U.S. Pharmacopeia, MD(2001)).

Example 4 Otitis Externa—Animal Studies

Representative solutions of the instant invention were tested as apretreatment for the manifestations of otitis externa prior to othermedication in 50 animals, mostly dogs, ranging from 6 months to 15 yearsof age. The solution was instilled into the ear canals followed bygentle massage. Instillation and massage occurred 10-15 minutes prior toany additional medication. The manifestations of otitis externa wereattributed to food allergies, recurrent Malassezia otitis,staphylococcus/streptococcus/pseudomonas otitis, otitis with exudate,pruritus, erythematous otitis, atopy, ear mites, ruptured tympanicmembrane, excoriation, ulceration, chronic recurrent otitis withhyperplasia, hematoma, and pemphigous foliaceus.

No irritation was reported in 49 of the 50 animals examined (one animallost to study). In four of the 50 animals tested, the solution of theinstant invention was the sole medication. Of these four animals, threehad 100% resolution of the symptoms of otitis externa following 14 daysof treatment. The remaining animal had a 50% resolution of symptoms whenthe owner removed the animal from the study.

Complete resolution of symptomology occurred when the instant inventionwas used in conjunction with the following commonly used agents:Zeniquin (marbofloxacin), Clovamox (amoxycillin trihydrate/clavulanatepotassium), hydrocortison, prednisone, Tresaderm(thiabendazole-dexamethasone-neomycin sulfate solution),neomycin/polymixin drops, Otomax (gentamicin sulfate, betamethasonevakerate, clotrimazole), Baytril Otic (enroflozacin/silversulfadiazine), Conofite (miconazole nitrate), and Gentocin otic(gentamicin sulfate/betamethasone valerate).

Example 5 Antimicrobial Properties

To illustrate the antimicrobial properties of representative solutionsof the invention, the resolution of the bacterial infections associatedwith the otitis externa for the four animals treated only with therepresentative solution of the invention (see Example 4) are describedherein.

Sub- Microbiology Treat- Resolution ject Type History at Onset ment (%) 7 Mixed K-9 Ear mites Gram (+) 14 days 100*  cocci culture S.intermedius  9 Dalmatian Atopic Gram (+) 14 days 100*  dermatitis cocciculture 10 English Pruritic skin Gram (+) 10 days 100*  Mastiff diseasecocci culture 13 Cocker Invasive Gram (+) 21 days  50** Spaniel lymphomacocci culture & Malassezia *= Negative bacterial cytology at cessationof treatment and 1 week post treatment **= Cocci still present whenanimal removed from study by owner

Example 6 Antimicrobial Properties

The antimicrobial activity of the commercial product T8 Solution™prepared as per Example 1, (DVM Pharmaceuticals, Inc., Miami, Fla.) wascompared versus the antimicrobial activity of a T8 Solution placebo andthe commercial product TRIZ EDTA™ (533 mg TRIS, 141 mg EDTA, 112 ml H₂O,buffered to pH 8.0, DermaPet, Inc. Potomac, Md.). The antimicrobialactivity was evaluated against eight organisms Escherichia coli, Candidaalbicans, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiellaoxytoca, Proteus mirabilis, Proteus vulgaris and Staphylococcusintermedius.

Test organisms were obtained from the American Type Culture Collection(ATCC), Manassas, Va. and stored at low temperature (−70° C.) untilused. The test included eight microbial strains: Candida albicansATCC10231, Pseudomonas aeruginosa ATCC 9027, Proteus mirabilis ATCC33583, Proteus vulgaris ATCC 6383, Escherichia coli ATCC 8739,Staphylococcus aureus ATCC 6538, Staphylococcus intermedius ATCC 29663and Klebsiella oxytoca ATCC 49473. Each organism was evaluatedseparately as opposed to a mixed culture inoculum evaluation.

Stock cultures were maintained on Soybean-Casein Digest Agar (SCDA)slants for bacteria and Sabouraud Dextrose Agar (SDA) slants for fungi.Test cultures were prepared either on SCDA plate (bacteria) or SDA plate(fungi) as per USP 51 methodology, harvested, and adjusted to about 10⁸microorganism per ml for each test culture.

The number of colony-forming units (cfu) per ml in each microbialsuspension was determined immediately using standard plate-counttechniques, appropriate media, incubation time and temperature ((USPTest Method 61, U.S. Pharmacopeia 25, U.S. Pharmacopeia, MD (2001)).This value served to determine the inoculum size used for the test.

A normal saline solution (0.9% sodium chloride) was used as suspendingand diluting fluid for bacterial and yeast cultures. A Soybean-CaseinDigest Broth supplemented with 4.0% Polysorbate-20 and 1% Lecithin wasused as product diluent. SCDA was used as culture media for bacterialcount and SDA was used as culture media for fungi count.

Three samples (products) were tested for the study, T8 Solution™ (LotV2C020A, DVM Pharmaceuticals, Inc. Miami, Fla.), T8 Solution Placebo(Lot 940-19D, DVM Pharmaceuticals, Inc. Miami, Fla.), and Triz EDTA™(Lot 21068, DermaPet, Inc. Potomac, Md.). Both T8 Solution™ and the T8Solution placebo were used as is. The T8 Solution placebo does notcontain benzyl alcohol. Triz EDTA™ was reconstituted as per the labelinstructions before use.

Test samples were inoculated with a suspension (no more than 0.1 ml) ofone of the test organisms to give a final microbial concentration ofabout 10⁵ to about 10⁶ microorganisms per ml of the preparation. Thesamples were mixed thoroughly to ensure homogeneous distribution. Theinoculated samples were incubated at 22.5±2.5° C., and protected fromlight for 42 days.

Samples were removed from each tube at 24, 72 hours and after 7, 14, 21,28 and 42 day incubation and the number of viable microorganismsdetermined using the standard plate count technique.

The USP requirement antimicrobial effectiveness for bacteria is “No lessthan 2.0 log reduction from the initial count at 14 days, and noincrease from the 14 days' count at 28 days and for fungi: “No increasefrom the initial calculated count at 14 and 28 days.”

Both T8 Solution™ and T8 Solution placebo met the USP 26 Test 51Antimicrobial Effectiveness Testing for all 8 microorganisms tested.

Under the same experimental conditions, Triz-EDTA™ failed the USPtesting for S. aureus and Klebsiella oxytoca. Triz EDTA™ failed to meetthe “two log reduction within 14 days” requirement. (less than 1% ofinitial counts). Triz EDTATM did meet the USP 26 Test 51 AntimicrobialEffectiveness test standard for E. coli, S, intermedius, P. aeruginosa,C. albicans, Proteus mirabilis and Proteus vulgaris.

The rate of microbial count reduction for T8 Solution™ was significantlygreater than the T8 Solution placebo or Triz-EDTA™. E. coli, P.aeruginosa, Klebsiella oxytoca, Proteus mirabilis and Proteus vulgariswere reduced by T8 Solution™ to a non-detectable level (<10 cfu/g,colony forming units per gram of sample) within 24 hours. Both S. aureusand S. intermedius were reduced by T8 Solution™ to a non-detectablelevel within 72 hours. Candida albicans was reduced by T8 Solution™ toless than one percent of initial counts within 24 hours and tonon-detectable level within 14 days.

The number of viable microorganisms initially present at the start ofthe tests and the number of viable microorganisms detected at thespecified intervals, for the eight test organisms are given in TablesI-VIII.

TABLE I Number of viable microorganisms present at the indicated timeintervals (cfu/ml). Organism: Escherichia coli 24 72 7 14 21 28 42Inoculum hrs hrs days days days days days T8 580,000 <10 <10 <10 <10 <10<10 <10 T8 placebo 580,000 4300 5 <10 <10 <10 <10 <10 Triz EDTA 580,0007250 350 100 <10 <10 <10 <10

TABLE II Number of viable microorganisms present at the indicated timeintervals (cfu/m1). Organism: Ps. aeruginosa 24 72 7 14 21 28 42Inoculum hrs hrs days days days days days T8 150,000 <10 <10 <10 <10 <10<10 <10 T8 placebo 150,000 <10 <10 <10 <10 <10 <10 <10 Triz EDTA 150,000<10 <10 <10 <10 <10 <10 <10

Table III Number of viable microorganisms present at the indicated timeintervals (cfu/m1). Organism: S. aureus Inoculum 24 hrs 72 hrs 7 days 14days 21 days 28 days 42 days T8 345,000 100 <10 <10 <10 <10 <10 <10 T8placebo 345,000 298,000 265,000 116,500 1350 <100 <10 <10 Triz EDTA345,000 238,000 222,500 192,500 185,000 <100 <10 <10

TABLE IV Number of viable microorganisms present at the indicated timeintervals (cfu/ml). Organism: Klebsiella oxytoca Inoculum 24 hrs 72 hrs7 days 14 days 21 days 28 days 42 days T8 265,000 <10 <10 <10 <10 <10<10 <10 T8 placebo 265,000 20 10 <10 <10 <10 <10 <10 Triz EDTA 265,00073,500 67,500 56,000 19,500 2050 140 <10

TABLE V Number of viable microorganisms present at the indicated timeintervals (cfu/ml) Organism: Proteus mirabilis Inoculum 24 hrs 72 hrs 7days 14 days 21 days 28 days 42 days T8 620,000 <10 <10 <10 <10 <10 <10<10 T8 placebo 620,000 26,500 600 400 5 <10 <10 <10 Triz EDTA 620,00043,000 15,000 8,500 7,500 800 600 <10

TABLE VI Number of viable microorganisms present at the indicated timeintervals (cfu/ml). Organism: Proteus vulgaris Inoculum 24 hrs 72 hrs 7days 14 days 21 days 28 days 42 days T8 455,000 <10 <10 <10 <10 <10 <10<10 T8 placebo 455,000 3050 <100 <10 <10 <10 <10 <10 Triz EDTA 455,00024,000 300 40 <10 <10 <10 <10

TABLE VII Number of viable microorganisms present at the indicated timeintervals (cfu/ml). Organism: Staph, intermedius Inoculum 24 hrs 72 hrs7 days 14 days 21 days 28 days 42 days T8 240,000 2050 <10 <10 <10 <10<10 <10 T8 placebo 240,000 316,000 118,500 102,500 <100 <10 <10 <10 TrizEDTA 240,000 348,000 334,000 297,500 1300 <10 <10 <10

TABLE VIII Number of viable microorganisms present at the indicated timeintervals (cfu/ml). Organism: C. albicans Inoculum 24 hrs 72 hrs 7 days14 days 21 days 28 days 42 days T8 610,000 3550 1200 25 <10 <10 <10 <10T8 placebo 610,000 145,500 101,000 41,000 3400 800 190 20 Triz EDTA610,000 138,500 130,000 41,500 1950 375 45 <10

EQUIVALENTS

While this invention has been particularly shown and described withreferences to embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the spirit and scope of the invention asdefined by the appended claims. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedspecifically herein. Such equivalents are intended to be encompassed inthe scope of the appended claims.

1. A method of washing a wound comprising washing the wound with anaqueous antimicrobial and preservative alkaline solution, said solutioncomprising: (a) buffer system for maintaining the solution at analkaline pH; (b) surfactant system; (c) metal ion chelating agent; and(d) a non-ionic preservative having antimicrobial activity, wherein theaqueous antimicrobial and preservative alkaline solution isnon-irritating.
 2. The method of claim 1, wherein the buffer system isselected from the group consisting of a Tris buffer system, a phosphatebuffer system, and a citrate buffer system.
 3. The method of claim 1,wherein the buffer system is a Tris buffer system comprisingtromethamine base at a concentration of about 0.3 to about 0.55% (w/w)and tromethamine hydrochloride at a concentration of about 0.3 to about0.45% (w/w).
 4. The method of claim 1, wherein the pH is from about 8 toabout 9.5.
 5. The method of claim 1, wherein the surfactants arenon-ionic surfactants.
 6. The method of claim 1, wherein the surfactantis selected from the group consisting of nonoxynol 12, PPG-12/PEG-50lanolin, phospholipid CDM, phospholipid EFA, phospholipid PTA, PEG-100stearate, and any other nonoxynol, octoxynol surfactants with differentPEG chain lengths.
 7. The method of claim 1, wherein the metal ionchelating agent is selected from the group consisting of citric acid;phosphates; disodium EDTA; tetrasodium EDTA; ethyleneglycol-bis-(b-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA);1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA);ethylene-N,N′-diglycine (EDDA); 2,2′-(ethylendiimino)-dibutyric acid(EBDA); lauroyl EDTA; dilauroyl EDTA, triethylene tetraminedihydrochloride (TRIEN), diethylenetriamin-pentaacetic acid (DPTA),triethylenetetramine hexaacetic acid (TTG), deferoxamine, Dimercaprol,zinc citrate, penicilamine succimer, Editronate, and edetate calciumdisodium.
 8. The method of claim 1, wherein the preservative is selectedfrom the group consisting of imidazolindyl urea, benzalkonium chloride,diazolindinyl urea, dimethylol dimethylhydantoin, mixtures of5-chloro-2-methyl-4-isothiazolin-3-one and2-methyl-4-isothiazolin-3-one, benzyl alcohol, phenylethyl alcohol,phospholid CDM, and chlorobutanol, parabens, benzoic acid, and sorbicacid.
 9. The method of claim 1, wherein the preservative is benzylalcohol.
 10. The method of claim 4, wherein the surfactants arenon-ionic surfactants.
 11. The method of claim 4, wherein thepreservative is benzyl alcohol.
 12. A method of treating a topicalmicrobial infection of a tissue comprising washing the tissue with anaqueous antimicrobial and preservative alkaline solution, said solutioncomprising: (a) buffer system for maintaining the solution at analkaline pH; (b) surfactant system; (c) metal ion chelating agent; and(d) a non-ionic preservative having antimicrobial activity, wherein theaqueous antimicrobial and preservative alkaline solution isnon-irritating.
 13. The method of claim 12, wherein the pH is from about8 to about 9.5.
 14. The method of claim 12, wherein the surfactants arenon-ionic surfactants.
 15. The method of claim 12, wherein thepreservative is benzyl alcohol.
 16. A method of preparing a tissue forsurgery comprising washing the tissue prior to the surgery with anaqueous antimicrobial and preservative alkaline solution, said solutioncomprising: (a) buffer system for maintaining the solution at analkaline pH; (b) surfactant system; (c) metal ion chelating agent; and(d) a non-ionic preservative having antimicrobial activity, wherein theaqueous antimicrobial and preservative alkaline solution isnon-irritating.
 17. The method of claim 16, wherein the pH is from about8 to about 9.5.
 18. The method of claim 16, wherein the surfactants arenon-ionic surfactants.
 19. The method of claim 16, wherein thepreservative is benzyl alcohol.
 20. A method of topically delivering apharmaceutical agent to a tissue comprising: (a) mixing thepharmaceutical with an aqueous antimicrobial and preservative alkalinesolution, said solution comprising: (i) buffer system for maintainingthe solution at an alkaline pH; (ii) surfactant system; (iii) metal ionchelating agent; and (iv) a non-ionic preservative having antimicrobialactivity, wherein the aqueous antimicrobial and preservative alkalinesolution is non-irritating; and (b) applying the resulting mixture tothe tissue.